The long-term goal of this project is to use mouse models of transplantation to address focused and mechanistic questions about requirements for, and barriers to, tolerance, and to use this knowledge to develop new approaches to induce reproducible and durable graft acceptance. In this renewal application, we are continuing to focus on novel mechanisms of resistance to transplantation tolerance. We are particularly interested in two pathways, CD28 and the IL-17/T17 pathway, each of which have critical roles in alloreactivity and in ultimately dictating the phenotype of the alloimmune response, and thus the fate of the allograft. Using both wild-type and T cell receptor transgenic mice in combination with fluorochrome-reporter and gene-targeted mice, we will study how manipulations of these pathways influence the generation and homeostatic maintenance of effector/memory and regulatory T cells. These experiments particularly will address our central concept, which is that our understanding of how to manipulate the CD28 and T17 pathways to minimize effector responses, and optimize regulation, is incomplete. Thus, our goal is to better understand these pathways in order to develop novel approaches to more effectively target them and induce tolerance. To accomplish these goals we propose three aims: In Specific Aim #1, we will test the hypothesis that blockade of CD28 costimulation has differential effects on allograft responses depending upon whether the effector arm or the regulatory arm predominates. Using combinations of physiologic MHC mismatched models, foxp3 reporter mice, and TCR transgenic mice which allow for cell fate tracking, we will test differential requirements for CD28 signals in effector and regulatory-dependent responses. In Specific Aim #2, we will use conditional targeting of CD28 to study its role in effector and regulatory T cells. We have just completed the construction of CD28-floxed mice, which have now gone germline. Through the use these CD28-floxed mice we will delete CD28 in all T cells, Tregs (using foxp3-Cre mice) under temporal control with tamoxifen (using CreT2 mice). We will specifically test hypotheses regarding the roles of CD28 in Tregs, and at different time points in the allograft response. Lastly, in Specific Aim #3, we will study two aspects of T17 biology relevant to transplantation. First, we will test the hypothesis that T17 cells participate in allograft rejection by promoting Tc and Th1 responses, and second, we will investigate the role of CD28 (using CD28-conditional mice) and the novel Tim-1 pathway (using blocking Abs) in promoting T17 responses. Together these studies should provide important new insights into mechanisms of, and strategies for, tolerance.